The dramatic success of immune checkpoint blockade in treating patients with metastatic and/or locally recurrent melanoma has stimulated increasing efforts to investigate the application of this strategy to other solid tumors. Virus-associated solid tumors are ideal for evaluating immune resistance mechanisms, given the inherent inflammation associated with the local expression of foreign, highly immunogenic viral antigens. HPV-associated head and neck squamous cell carcinomas (HPV-HNSCC) originate in the tonsil, the major lymphoid organ that orchestrates immunity against oral infection. Despite its location in a lymphoid-rich environment, HPV escapes immune elimination during malignant transformation and progression. Our group recently showed that the immune checkpoint pathway, Programmed Cell Death-1 (PD-1) and Programmed Cell Death 1 Ligand-1 (PD-L1), facilitates persistent viral infection and subsequent development of cancer. Our findings support a model in which the PD-1:PD-L1 pathway becomes induced as an adaptive immune-resistance mechanism against the host tumor. We further investigated the immune signature of HPV-HNSCCs with activation of the PD-1:PD-L1 immune checkpoint pathway using a custom microarray with 59 immune-related genes. We found that IL-10 and IDO1 were the top two genes upregulated in PD-L1 expressing immune fronts. Interestingly, both of these genes are known to suppress host immune responses and both are targetable by drugs. Based on these findings, we propose to test whether blocking IL-10 and/or IDO1 pathways in HPV-HNSCC can enhance host anti-tumor immune responses. We propose to first quantify and geographically localize the expression of these proteins in a separate cohort of HPV-HNSCC patients using quantitative PCR and multiplex biomarker imaging and quantitative analysis. Subsequently, we will assess the efficacy of blocking in vivo either the IL10 and/or IDO1 pathway alone or in combination with anti-PD1 in a murine HPV tumor model. We then propose to correlate expression of these markers with clinical response to anti-PD-1 monotherapy in head and neck cancer patients. We hypothesize that expression of these genes can serve as a predictive biomarker and/or pathway of adaptive resistance to PD-1 blockade. The results may provide rationale for novel combinatorial therapies which target multiple immune checkpoints in future clinical trials. This proposal is in alignment with the NIH recognition of molecular signatures of tumor-host interactions, tumor microenvironments, and immune checkpoints as high priority areas of research.